CA1265811A

CA1265811A - O-alkyl platinum diamine compounds

Info

Publication number: CA1265811A
Application number: CA000515921A
Authority: CA
Inventors: Stephen Hanessian
Original assignee: Individual
Current assignee: Individual
Priority date: 1986-08-13
Filing date: 1986-08-13
Publication date: 1990-02-13
Also published as: CA1265811C

Abstract

ABSTRACT OF THE DISCLOSURE

This invention relates to compounds of the formula

Description

This invention relates, in one aspect; to platinum diamine compounds; in a further aspect, this invention relates to a method for preparing such compounds.
In a still further embodiment, this invention also relates to compositions containing, as the active inyredient, a platinum diamine compound.
Cis-Dichlorodiamineplatinum (also known as cls-platinum) is a dia~ine compound which is a first-1~ generation drug. The present-day knowledge concerning the structural, functional and geometrical requirements with platinum coordination compounds can be summarized by reference to the dichlorodiamamine platinum originally described by Rosenberg (B. Rosenberg et al., Nature 222, 385 (1969)~, as an example, and which is shown as both cis- and trans- isomers are known and~exist in aqueous solution in the 2+ oxidation state. Octahedral 4+ analogs with loosely bound ligands are also known, but the main emphasis has been on the Pt(II) compounds. A
most relevant finding is that only the cls-analog exhibits certain activity, the trans being very weakly active if at all. This reflects on the modes of binding to DNA. Cis-Pt(II) is a bifunctional reagent with square planar geometry in which the chloride ligands are prone ~6~

to substitution by incoming groups that are either in large excess (water) or form thermodynamically more stable links to platinum tDNA~. The amine groups are both kinetically and thermodynamically inert to substitution, but Eor their use as pharmaceutical components, they must play a key role in secondary binding to biological receptors such as DNA by H-bonding forces. The nature of the amine has an influence on the activity and possibly toxicity. For example, maximum activity and markedly reduced toxicity is observed with the nbutylamine derivative~ Tertiary amine derivatives are inactive. Low solubility still poses a major problem in intravenous administration of these drugs when cls P5(II) is dissolved in water, the two chloride ligands are sequentially exchanged for water or hydroxy depending on the pH. At 37 the half-life for the completion of this reaction is 1.7h with an activation enthalpy of about 20 Kcal/mol. The aquated forms of cls-Pt(II) appear to be important bioactive species as they would be 0 expected to exist inside the cell, because of a favourable pH balance.
With the present invention, applicant has developed novel compounds which are described in greater detail hereinafter, but which are of the general formula X \ ~ N - CH
Pt \
X N - CH

wherein Rl and R2 are independently selected from alkyl groups comprising n carbon atoms, wherein n equals 1 - 8 containing at least one lower alkoxy group of 1 - 3 carbon atoms, and X is a protective group, e.g., a halogen.
In accordance with another aspect of the present invention, there is provided a process of preparation of a compound of the general formula R

X ~ N --- CH

Pt ~ \
X N - T ( ~H

wherein Rl and R2 are independently selected from a carbon chain comprising n carbon atoms wherein n equals 1 - 8 containing at least one lower alkoxy group of 1 - 3 carbon atoms, and X is a protective group, e.g~, a halogen, comprising reacting a compound of the formula NH2 -- CE~ -- Rl NH2 ~ CH - R2 ~26~5~

wherein R1 and R2 are defined as above, with K2PtX4, wherein x is defined above. ln the above ~ormulae, X may also be a mono- or dicarboxylic acid; suitable acidic ligands or aqua groups may also be employed.
In greater detail of the process aspect, for the preparation of the platinum coordination complexes several methods can be used. The starting material may be a potassium tetrachloroplatina-te (II) (K2PtC14). To a clear solution of this salt is added directly the l() organic-amine ligand in water in alcohol, and allowed to stand at room temperature or heated up to 65C until the color is changed from red-blood to orange-yellow or blue-green (in some cases). This then is followed by standard pocedures of separation, purification, identi~ication and chemical analysis. Separation of the various complexes is done also by chromatography and high pressure liquid chromatography (both analytic and preparative). The latter technique would appear to be a particularly potent tool to investigate the reactions of platinum coordination compounds with nucleotides and nucleic acids.
The diamino compounds described herein can also be prepared by other methods. Thus within each series, the diamine derivative could be prepared by displacing suitably protec-ted derivatives ~sulfonates, halides) with azide ion or ammonia. This could be done on 4,5 and 6-carbon acyclic or cyclic derivatives (such as~lycosides). In the latter case, the glycosides could be hydroly~ed and reduced to -the corresponding alditols.
The methyl ethers can be formed prior to the introduction of the amines or after (in case of azides). Platinum complexes can also be preparea from the diamino compounds in a conven~ional manner if desired. Epoxides can also be used as starting materials for the preparation of the diazides or the diamines.
In greater detail of the present invention, the compounds of the above formula have been found to possess antimicrobial activity, making them suitable for use as antimicrobial agents which can be combined with pharamceutically acceptable carriers to form antimicrobial compositions.
In addition to the above, the platinum diamine compounds of the present inven-tion also have been Eound to possess activity against cer-tain strains of certain types of tumors. In this respect, the known cisplatinum compound is, of itself, in a unique class of coordination compounds, that has therapeutic activity against a wide spectrum of human tumors, either alone or in combination with other agents such as adriamycin. In fact, recently the FDA approved cis-platinum Eor use in humans.

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Although the mode of action is still unknown, it has been found that cis-platinum and its analogs bind to the DNA
and disturb the normal functions of the cell. In another hypothesis, the drug is believed to enhance antigenicity so that tumors become more susceptible to destruc-tion by the host's immune ~ystem. Regardless of the precise mode of action, the efficacy and potential of cis-platinum as an antitumor agent are of some significance. Although cis-platinum exerts its preferential toxicity to tumor 1~ cells when compared to normal cells at a therapeutic index which allows its clinical use, the situation is still far from perfect and there are also serious side effects to overcome. An improvernent is stil] sought in therapeutic index (presently 2-2,5 mg/Kg). Among the major drawbacks are nephrotoxicity, gastro-intestinal problems and depression of the function of the bone marrow thu~ producing fewer than normal white blood cells and platelets. These have obviously limited the size of the dose and continue to be a major impediment to the routine use of cis-platinum in humans.
Presently, patients are induced to increase their flow of urine by intravenous administration of 1-2 liters of fluid. The drug is then administered intravenously together with a diuretic. A most recent development, is the administra~ion of D-mannitol which is believed to "flush out'l residual cisplatinum from the body by coordination. These are nonetheless relati~ely harsh treatments even in the face of life-threatening situations.
There is at present a renewed interest in the treatment of a variety of tumors with platinum coordination compounds. (B. Rosenberg, Cancer treatment reports 68, 1343 (1979); and Science, 192, 774 (1976;

Chem. Eng. News, Jan. 21, 1980, p.35)~ Reports on the clinical status of clsPt(II) in cancer chemotherapy, generally on extremely advanced cases, indicate promising anticancer activity. (J. A. Gottieb and B. Drewinko, Cancer Chemother. Rep., 59, 621 (1975)). For example, researchers at Georgetown University in Washington have reported remarkable success in treating brain tumors with this drug (Chem. Eng. News, Oc-t. 6, 1980, p. 27). The patients in this case were 10 children whose tumors had resisted many forms of chemotherapy. Cis-Pt(II)~ alone 0 or ln combination has had success in the treatment of testicular carcinoma (~. J. Wallace and D. J. ~igby, Recent Results Cancer Res., 48, 167 (1974)), hand and neck cancer (I. H. Krakoff and A. J. Lippman, Recent Results Cancer Res., 48~ 183 (1974)), squamous cell ~2~

carcinoma, malignan-t lymphoma and endometrial carcinoma (J. M. Hill et al, Cancer Chemother. Rep., 48, 145 (1974) and J. M. ~lill et al., Cancer Chemother. Rep., 59 647 (1975), and ovarian adenocarcinoma (~. Wittshaw and B. Care, Cancer Res., 48, 178 (1974).
As previously mentioned, cis Pt(II) treatment has certain disadvantages. For example the ir is not very soluble in water which renders its intravenous administration problematic Some derivatives are known 1~ to maintain the desired levels of activity (T. A.
Connors, M.J. Cleare and K.R. Harrap, Cancer Treatment REports 63, 1499 (1979)), but the solubility characteristics were not noticeably improved; thus, e.g., malonato (1,2-diaminocyclohexane) platinum (II) has been found to be effective against a number of animal tumors in addition to L1210 Leukemia (J. H.
Burchenal et al., Cancer Treatment Reports, 63, 1493 (1979)). Nephrotoxicity and other forms of toxicity remain as the most serious drawback of c1s-Pt(II) and the limited number of analogs tested so far. The synthesis of different platinum coordination compounds with greater water solubility, less renal toxicity and greater antitumor activity is a goal that is still actively sought by investigators tsee Roberts and Thomson below).

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The primary mode of action (J. J. Roberts and A.
J. Thomson, Prog. Nucl. Acid Res., Mol. Biol., 22, 71 (1979) and A. D. Kelman and H. J. Persesie, Cancer Treatment Repor-ts, 63, 1445 (1979)) of cis-Pt(II) and its derivatives is localized at the intracellular DNA level.
While the sites within the various bases to which cls-Pt(II) binds and their relative importance to the antitumor activity have not been conclusively discerned, there is good evidence that DNA interstrand (A. D. Kelman 1~ and H. J. Persesie, Cancer Treatment Reports, 63, 1445 (1979)) cross-linking, most probably at adjacent guanine pairs, or interstrand crosslinking (P. K. Ganguli and T.
Theophanides, Euchem Conference on Coordination Chemistry and Cancer Chemotherapy, Toulouse, France, 1978; and Eur.
J. Biochem 101, 377 (1979)) at N(7) and C (6)-0 on guanine may be responsible. The bou~d cls-Pt(II) drug would result in template inactivation of DNA and the accumulation of potentially lethal damage.

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With respect to their transport, again, little is known except that the drug mu.st be able to penetrate the cell membrane prior to exerting intra-cellular tumoridical action. The active form must be electrically neutral in the plasma prior to transport. Otherwise a carrier-mediated mechanism has to be involved for which there is no evidence (L. ~. Zwelling and K. W. Kohn, Cancer Treatment Reports, 63, 1439 (1979)).
The new compounds of the present invention have several advantages over known compounds, namely:
1. Superior water solubility

2. When used as pharmaceuticals, they have more efficient exclusion Erom the body

3. Improved transport (maintaining charge neutrality)

4. Possible oral activity

5. Possible different metabolism

6. Better balance oE hydrophobic and hydrophilic character ~more eEfective transport and distribution in plasma).

7. Interference with repair en~ymes which reverse the drug-inflicted damage by an excision repair process

8. Improved biocompatibility in plasma and other fluids ~5~ L

9. Inherent chirality (selective interaction with certain receptors, membrane constituents and the DNA
helix itself).
In addition, the chiral, functional nature oE the analogs may increase their effectiveness due to:
1. Added binding sites to the DN~ and intracellular proteins 2. Presence oE auxiliary stabilizing structures telectrotatic stabili~ation by polar groups) ln 3. Presence oE masked aquasystems (physiologically active form of cis-platinum.
The compositions oE the present invention having use as anti-microbial compositions or for the treatment of tumors may be formulated using conventional techniques so as to include pharmaceutically acceptable carriers or diluents.
The compositions may be formulated as, e.g., powders, solutions, or suspensions which have been conventionally produced using acceptable diluents. The ~Q compositions may also be formulated for parenteral administration by injection or continuous infusion when used to treat tumours; such formulations for injection may be present in unit dose form as ampoules in multiple-dose contai~ers with added preservative.

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- -The compositions may also assume such forms as suspensions, solutions or emulsions in oily or aqueous carriers and may contain Eormulation aids, such as suspending agents, stabilizers and/or dispersants.
~ lternatively, the active principle may even be present in powder form Eor reconstitution before use with a suitable carrier, for example, sterile, pyrogen-free ~ater.

Having thus generally described the invention,

10 reference will now be made to the accompanying examples, illustrating preferred procedures.

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EXI~MPLE
l~2:5l6-Di-o-Isopropylidene-3~4-d_O-methanesulfonyl-D-mannitol A solution of 1,2:5,6-di-O-isopropylidene-D-mannitol (8.4g, 32mmoles) in dry methylene chloride (144mL) and triethylamine (llg,O.lmmole) is treated with methanesulfonyl chloride (8.9g, 0.008mmole) dropwise at OC under nitrogen atmosphere. After allowing the mixture to warm up to room temperature, it is washed ln abundantly with cold distilled water and the organic phase is dried tsodium sulfate) and evaporated under vacuo, to afford (yield 12g; 90%) of a colorless crystalline product. M.P. 142-143C [~]D = -5, (C=l, CHC13). For the preparation of the mannitol starting compound, see E. Baer, Biochemical Preparations, 2, 31 1952 ).

3,4-Diazido-3,4-dideoxy-1,2:5,6-di-O-isopropylidene-D-iditol To a solution of 1,2:5,6-l~i-O-Isopropylidene-3, 4-di-O-methanesulfonyl-D-mannitol (12g, 28.7mmoles) in dry toluene (300mL) is added tetrabutylammonium azide t24g, 3eq.). The mixture is heated at reflux temperature under stirring for 12 hours, poured into ~, i8~

water t500mL) and extracted. TiIe organic extract is washed with brine, dried (sodium sulfate), and evapor~ted at 50C. Chromatography on silica gel (3:1 hexane-AcOEt) afEorded a pale yellow syrup (yield:
3.59g, 40~). Sublimation at 110C (0.3mm Hg) gave the iditol crystalline product which melts at room temp.
[~D25 = ~ 130~ (C,l, CIIC13).
The deprotection of 3,4-Diazido-3,4-dideoxy-1,2:5, 6-di-O-isopropylidene-D-iditol was executed under 1~ mild conditions (AcOH 120 ml, H20 60 ml, at 70C for 2 hours) to afford -the crystalline diazidopolyol derivative.

3,4-Diazido-3,4-dideoxy-1,2:5,6-tetra-O-methyl-D-iditol ~ solution of the preceding diazido polyol (500mg, 2.15mmoles) in dry dimethylformamide t7.5mL) is transferred to a round bottom flask containing sodium hydride t60~ oil dispersion, 0.688 g, 8eq.) in dry dimethylformamide t5mL) at OC under nitrogen ~tmosphere. Upon ceasing of effervescence ( 45min), methyl iodide (3.05g, 10 equiv.) is added dropwise. The reaction mixture is then stirred at OC for 1 hr., and left standing at r~om temperature overnight. The mixture is neutralized with methanol followed by N
sulfuric acid. A large amount of ~,~

~Z658 saturated sodium chlo~ide solution is added to the mixture, and then ~he mixture is extracted with ether.
The organic extract is dried (sodium sulfate), evaporated unaer ~acuum and the resultiny syrup chromatographed on silica gel (35:65 ethyl acetate-hexane) to afEord the pure product as a colorless syrup; (yield: 554mg, 89%, [~D25 = 188 (C=l, C~IC13).

3-4-Viamino-3,4-dideoxy-D-iditol To a solution oE the diazido polyol in Example 2 (406 mg, 1.75 mmole) in methanol (30mL) and distilled water (5mL) is added 10% palladium-on-charcoal (150mg) suspended in water (5mL). The mixture is hydrogenated under atmospheric pressure at room temperature for 24 hours (an infrared oE an aliquot is taken at this time to insure disappearance oE the azide band). The catalyst is then removed by filtration through celite, washed with distilled water (5mL), and the filtrate evaporated to dryness by co-distillation with absolute ethanol. A white solid is obtained (yield 302mg, 85%);
recrystallization from hot ethanol/water aEEorded colorless needles (yield of recrystallization: 50%).
M P 160-161C, [~]D25 = -104 (C=l, H20).

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2,3-Diazido-2,3-dideox~-D-threitol -To a solution of the diazido polyol (Example 2) (500mg, 2.15mmoles) in distilled water (lOmL) is added sodium metaperiodate (92mg, 4.32mmoles, solid), portionwise at OC, in the absence of light, the mixture is stirred Eor 2 hours at oc. then tre~ted with ~odium borohydride (eq.) in distilled water (lOml). After two hours, the reaction is neutralized with N sulfuric acid, 10 decolorized with sodium thiosulfate and evaporated under vacuum. The residue is dissolved in water (25mL) and extracted with ethyl acetate (5 x 40 mL); the combined organic extracts is decolorized with sodium thiosulfate, dried and evaporated under vacuum to afford a colorless oil (390mg, 96%). Thin layer chromatography of the oil shows the presence of the diol 2,3-Diazido-2,3-dideoxy-D-threitol and the triol 2,3-Diazido-2,3-dideoxy-D-xylitol in e~ual proportion. The oil is recycled through the oxidative cleavage and the reduction as ~ described above and the reaction mixture is processed similarly to afford the diol 2r3-Diazido-2,3-dideoxy-D-threitol in the form of white crystals;
recrystallization in chloroEorm/hexane yields 303mg, (82%~ of product. M.P. 78-79C, [~]D = ~41.4 (C=0.5,Et20).

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2,3-Diazido-2,3-dideoxy-D-XylitOl To a solution of the diazido polyol (Example 3) ~lg, 4.3mmol) in distilled water (25mL) is added sodium metaperiodate (920mg, leq) in distilled water (lOmL), portionwise at OC, in the absence of light. The mixture is stirred 1 1/2 hours at OC, then treated with s~dium boroh~dride (3 e~.) in distilled water (20mL).
~Eter 1/2 hour, the reaction is neutralized with I
sulfuric acid. (The mixture may be decolorized with a few milliliters of saturated sodium thiosulfate), and evaporated under vacuum. The residue is dissolved in water (25mL) and extracted with ethyl acetate (5 x lOOmL). The combined organic extract is decolorized with sodium thiosulfate, dried, evaporated under vacuum and chromatographed on Silica gel (85:15 ethyl acetate-hexane) to afford the diol namely 2,3-Diazido-2,3-dideoxy-D-threitol (69mg, 10%), and the triol, namely 2,3-Diazido-2,3-dideoxy-D-xylitol (577mg;
66~ yield). [~]D 5 = -239 (C=1.35, CH3QH), IR ma~:
2100cm 1 (N3).

General Methylation Procedure A solution of polyol (2.15rrlmoles) in dry dimethyl formamide (75m~) is transferred to a round bottorn flask - 1,8 -containillg sodium hydride (20 equiv) in dry dimethyl formamide (5mL) at OC under nitrogen atmosphere. Upon ceasing of effervescence (45 min) methyl iodide (10 equiv) is added dropwise. The reaction mixture is stirred at o~C for 1 hour, and le~t standing at room temperature overnight. After the reaction is finished, the mixture is neutralizeA with methanol Eollowed by N
sul~uric acid. The organic phase is processed as usual to give a syrup. Chromatography on silica gel using

11) 35:65 v/v ethyl acetate-hexane as the eluant gives the pure desired product. Ethylation was performed with ethyl iodide.

2,3-diamino-2,3-dideoxy-1,4-di-O-ethyl-D-threitol, 2,3-diamino-2,3-dideoxy-1,4-di-O-methyl-D-threitol, 2,3-diamino-2,3-dideoxy-1:4,5-tri-O-methyl-D-xylitol and 3,4-diamino-3,4-dideoxy-1,2:5,6-tetra-O-methyl-D-iditol are prepared from the analogous diazido polyol by methylation following the process of Example ~0 7, and subsequent reduction of diazido polymethyl ether using hydrogenation in the presence of palladium-on-charcoal.

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_s-Pt co~plex formation The complex formation of diamines 3,4-Diamino-3l4dideoxy-D-iditol~2~3-Diamino-2~3-dideoxy-D-threitol;
2,3-Diamino-2,3-dideoxy-D-xylitol, Methyl 2,3-dia~ino-2,3-dideoxy-~-D-allopyranoside and O-methyl analogs with tetrachloroplatinate are carried out according to the ~ollowing protocol; the substrate is dissolved in an a~lueous solution oE the platinum salt. The resulting homogeneous solution is left standing at room temperature in the absence of light, resulting in the slow crystallization of the desired complex. For example, the ligand 3,4-Diamino-3,4-dideoxy-D-iditol gives pure crystaline needles cis-~3,4-Diamino-3,4-dideoxy-D-iditol) dichloroplatinum ~II) whose structure was confirmed by single crystal X-ray crystallography.

2,3-Diazido-2,3-dideoxy-1:4-di-O-ethyl-D-threitol ~ solution of diol ~0.2g, 1.2mmoles) in dry 20 DMF(2.5ml) is transferred to a round bottom flask containing sodium hydride l60~ dislpersion, 4eq.) in dry ~MF(2.5ml) at OC under nitrogen atmosphere. Upon :,~

ceasing of eEfervescence (30min), ethyl iodide (0.4ml, ~eq.) is added dropwise. The reaction mixture is then stirred at 0C for 1 hrs, and left standing at roolr temperature overnight. A large amount of saturated soaium chloride solution is added and the mixture is extracted with ether. The organic phase is evaporated under ~vacuum. The resulting syrup is chromatographed on s,ilica ~el (~:1 Hexane: AcOEt) to aEford the pure product as a colorless syrup; yield: 0.2318g, 876.
1~ EXAMPLE 11 2,3-Diamino-2,3-dideox~-1,4-di-0-ethyl-D-threitol To a solution oE 2,3-diazido-2,3-dideoxy-1,4-di-0-ethyl-D-threitol(0.2318g, 1.0166mmoles) in methanol (5.5ml) is added palladium-on-activated-charcol(10%, Pd, lOOmg) suspended in distilled water(3.5ml). The mixture is hydrogenated under atmospheric pressure at room temperature for 24 hours. (An infrared of an aliquot is taken at this time to ensure disappearance of the azide). The catalyst is then removed by filtration ~0 through celite and evaporated under vacuum then coevaporated with ethanol to dryness to afford the product as a colorless syrup 0.1415g, 80% yield).

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- 2l -EX~MPL~ 12 cis-(2~3-Diamino-2~3-dideoxy-lr4-di-o-ekhyl-D-threitol)-dichloroplatinum( II ) 2,3-Diamino-2,3-dideoxy-1,4-di-0-ethyl-D-threitol(O.1415g) is transferred, using distilled water(O.3ml), into a solution containing K2PtC14(leq.) in distilled water(O.3ml). The mixture is stirred m~nually until homogeneous, and is left standing in the dar~. ~fter a few hours the slow crystallization process 1~ begins to give yellow needles. The crystals are Eiltered, washed with a little blt oE cold water and dried by suction of air, for several hours, to afford y~llow crystals (0.1612g, yield 45%).

cis-(2,3-Diamino-2,3-dideoxy-1,4,5-tri-0-methyl-D-xylitol)-dichloroplatinum(II) 2~3-Diamino-2~3-dideoxy-l~4~5-tri-o-methyl-D-~ylitol (2.6 g) is transferred, using distilled water (1~5 ml), into a solution containing K2PtC14 (leq., 5.6g) in distilled water (7 ml). The mixture is stirred manually until homogeneous, and is left standing in darkness. ~fter a few hours the slow crystallization i~

- 2~ -process begins in the Eorm of pale yellow fine crystals.
The crystals are filtered, washed with a little cold water, and dried under a suction of air Eor several hours to afford pale yellow fine crystals (2.5g, yield ~10~ ) .

cis-(2~3-Diamino-2,3-dideoxy~1~4-di-O-methyl-D-threitol)dichloroplatinum(II) To a solution of 2,3-diamino-2,3-dideoxy-1,4-di-O-methyl-D-threitol (0.7541 g) in distilled water (5 ml) was added K2PtC14 (1 eq., 2.1 g). The mixture is stirred manuaLly until homogeneous, and is left standing in darkness. After a few hours, the slow cr~stallization process begins in the form of yellow needles. The crystals are filtered, washed with a little cold water, and dried under a suction o~f air for several hours, the afford yellow crystals (1.2 g, yield 60%) '~

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EXAMPL~ 15 The product of Example 3 was hydrogenated in the manner oE the pre~eaing examples to yield the corresponding diamino compound; yield is 91~. [~]D25 =
-262 (in methanol). This was then converted to the corresponding dichloroplatinum complex compound by procedures described above to yield a product having a ~nelting point oE 286-287C. (with decomposition); [~]D 5 - -25.2 in water.

In addition to the compounds prepared above, the corresponding L-series can be prepared in an identical manner.

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- 2~ -Varlous of the compounds of the present invention were tested for antimicrobial activity. Testing was carrie~ out to determine the activity based on the growth of micro organisms tmeasured) and the mini~um inhibitory concentration determined Eor the respective compounds.
The method employed 96 well microtiter plates were prepared by adding 100 ~1 appropriate medium to each well. 50 ~1 aliquots of the diluted test compound are a~ded to each well in a manner resulting in the required dilutions and the proper controls. The test organisms are innoculated into each well in a volume of 10 ~1.
The trays are incubated at 37C for 16-24 hours for bacteria; yeast and fungi containing trays are incubated at 28 for 36-48 hours. The plates are read either with Dynatech MR 600 plate reader or manually.
In the following tables the indicated codès are used:
109050 is cis-dichlorodiamine platinum (II);
~0 114551 is cis-(3,4-diamino-3,4-dideoxy-1,2:5,6-tetra-O-methyl-D-iditol)dichloroplatinum (II);
115448 is cis-(2,3-diamino-2,3-dideoxy-1,4-di-O-methyl-D-threitol~dichloroplatinum (II);

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115449 is cis-(2r3-diamino-2~3-dideoxy-l~4~5-tri methyl-D-xylitol)dichloropl~tinum (II);
123597 is cis-(2,3-diamino-2~3-dideoxy-1,4-di-O-ethyl-D-threitol)dichloroplatinum (II).

TABLE I
~ntimicrobial ~Cti~Ji t~
Organism Salmonella Alcaligenes C~mpound Typhimurium Viscolachs lo 1145511000 333 The next series of tables (II, III, IV) exhibit ln vitro lack of cross resistance to strains sensitive (S) and resistant (R) to cisplatinum were tested against the compounds (L1210DPP is a resis-tant strain); t~he results are ~iven at ICso in Mg/ml tII), ID50 in uM (III) and ID50 in Mg/ml (IV).

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TABLE II
ICso (~g/ml) train _ ~ound L1210S L1210R
109050 0.21 0.53 109050~ 0.24 2.98 ~14551 15.14 15.60 1154~18 5.35 1.01 115~49 6.35 2.30 115449~ 7.36 6.98 # degree of resistance to cis platinum increased by second test.
The lack of cross resistance is noticeable.

TABLE III
ID50 (~M) Strain Compound L1210S L1210R
109050 0.8 9.9 114551 . 30.1 31.0 ~ 115449 16.5 15.7 123597 13.4 14.6 Again, the lack of cross resistance is apparent.

r~gLE IV
I D 5 0 ( ~I g/ml ) Strain Compound L1210S L1210R L1210DPPP388S P388R
109050Q.25 3.25 4.91 0.20 4.85 11455115.~ 15.6 - 27.9 27.0 115~8 5.35 11~4497.36 6.98 1235975.90 6.42 7.21 - 48.0 Lack of cross resistance shown to 114551 by L1210 and P388 strains, and 115449 and 123597 to L1210 strains.
The cells were incubated at 37C in a suitable medium te.g., RPM11640 supplemented with 10% fetal calf serum) with various concentrations of test age~t, and counted a~ter 72 hours for assessment.
Numerous in vivo tests were run against L1210 leukemia, M5076 sarcoma, and PV induced sarcoma (PV-239).
114551 had activity against L1210 intraperitoneal implants but less than 10~ of cis platinum, using intraperi~oneal treatment 115448 and 115449 had activity .~

~gail~st intraperitoneal implants of M5076 sarcoma using intraperitoneal treatment, but this was less active than cis platinum using intraperitoneal treatment.
114551 was not very active againt subcutaneous implants of M5076 sarcoma, which closely mimics the clinical situation.
11~551 and 115448 when tested against PV induced s~reoma were ineffective. 115449 was toxic to the subjects at all doses tested.
1~ 114551, 115449 were tested against L1210 and L1210/DPP in vivo, using intraperitoneal implants both showed better activity against L1210 than L1210/DPP.
Similar testing oE 115448 and 115449 against the same two strains under similar conditions gave similar results.
115448 when tested against P388 and P388/p~P showed better activity against P388 than P388/DPP.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A compound of the formula wherein R1 and R2 are independently selected from alkyl groups comprising n carbon atoms, wherein n equals 1 - 8 containing at least one lower alkoxy group of 1 - 3 carbon atoms, and X is a protective group.

2. A compound of the general formula wherein R is lower alkyl of 1 to 3 carbon atoms and m and p can be independently 0, 1, 2 or 3.

3. Cis-(2,3-diamino-2,3-dideoxy-1,4-di-O-ethyl-threitol)dichloroplatinum(II).

4. Cis-(2,3-diamino-2,3-dideoxy-1,4-di-O-methyl-threitol)dichloroplatinum(II).

5. Cis-(2,3-diamino-2,3-dideoxy-1:4,5-tri-O-methyl-xylitol)dichloroplatinum(II).

6. Cis-(3,4-diamino-3,4-dideoxy-1,2:5,6-tetra-O-methyl-iditol)dichloroplatinum(II).

7. A compound according to claim 3 or 4 wherein the compound has the D-form.

8. A compound according to claim 5 or 6 wherein the compound has the D-form.

9. A process of preparation of a compound of the general formula wherein R1 and R2 are independently selected from a carbon chain comprising n carbon atoms wherein n equals 1 - 8 containing at least one lower alkoxy group of 1 -3 carbon atoms, and X is a protective group comprising reacting a compound of the formula wherein R1 and R2 are defined as above, with K2PtX4, wherein X is defined above.

10. A process of preparation of a compound of the general formula wherein R is lower alkyl of 1 to 3 carbon atoms and m and p can be independently 0, 1, 2 or 3 comprising reacting a compound of the formula where R, m and p are defined as above, and K2PtCl4 in aqueous or alcoholic solution.

11. A process for preparation of cis-(2,3-diamino-2,3-dideoxy-1,4-di-O-ethyl-threitol)dichloroplatinum(II) comprising reacting 2,3-diamino-2,3-dideoxy-1,4-di-O-ethyl-threitol and potassium tetrachloroplatinate in aqueous or alcoholic solution.

12. A process of preparation of cis-(2,3-diamino-2,3-dideoxy-1,4-di-O-methyl-threitol)dichloroplatinum-(II) comprising reacting 2,3-diamino-2,3-dideoxy-1,4-di-O-methyl-threitol and potassium tetrachloroplatinate in aqueous or alcoholic solution.

13. A process of preparation of cis-(2,3-diamino-2,3-dideoxy-1:4,5-tri-O-methyl-xylitol)dichloro-platinum(II) comprising reacting 2,3-diamino-2,3 dideoxy-1,4,5-tri-O-methyl-xylitol and potassium tetrachloroplatinate in aqueous or alcoholic solution.

14. A process of preparation of cis-(3,4-diamino-3,4-dideoxy-1,2:5,6-tetra-O-methyl-iditol)dichloro-platinum(II) comprising reacting 3,4-diamino-3,4-dideoxy-1,2:5,6-tetra-O-methyl-iditol and potassium tetrachloroplatinate in aqueous or alcoholic solution.

15. A process as defined in claim 11 or 12 wherein the threitol reactant is in the D-form.

16 . A process as defined in claim 13 or 14 wherein the threitol reactant is in the D-form

17. A composition comprising a compound of the formula wherein R1 and R2 are independently selected from alkyl groups comprising n carbon atoms, wherein n equals 1 - 8 containing at least one lower alkoxy group of 1 - 3 carbon atoms, and X is a protective group, and a pharmaceutically acceptable carrier therefor.

18. A composition comprising a compound of the general formula wherein R is lower alkyl of 1 to 3 carbon atoms and m and p can be independently 0, 1, 2 or 3 and a pharmaceutically acceptable carrier therefor.

19. Cis-(2,3-diamino-2,3-dideoxy-1,4-di-O-ethyl-threitol)dichloroplatinum(II) and a pharmaceutically acceptable carrier therefor.

20. Cis-(2,3-diamino-2,3-dideoxy-1,4-di-O-methyl-threitol)dichloroplatinum(II) and a pharmaceutically acceptable carrier therefor.

21. Cis-(2,3-diamino-2,3-dideoxy-1:4,5-tri-O-methyl-xylitol)dichloroplatinum(II) and a pharmaceutically acceptable carrier therefor.

22. Cis-(3,4-diamino-3,4-dideoxy-1,2:5,6-tetra-O-methyl-iditol)dichloroplatinum(II) and a pharmaceutically acceptable carrier therefor.

23. A composition according to claim 19 or 20 wherein the compound has the D-form.

24. A composition according to claim 21 or 22 wherein the compound has the D-form.

25. A compound as defined in claim 1 wherein X is a mono- or dicarboxylic acid.

26. A process as defined in claim 9 wherein X is a mono- or dicarboxylic acid.

27. A composition as defined in claim 17 wherein X
is a mono- or dicarboxylic acid.